biomolecules Article Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren 1,2, 1,2, 1,2, 1,2 Sergio Castañeda-Zegarra y , Camilla Huse y, Øystein Røsand y, Antonio Sarno , Mengtan Xing 1,2, Raquel Gago-Fuentes 1,2, Qindong Zhang 1,2, Amin Alirezaylavasani 1,2, Julia Werner 1,2,3, Ping Ji 1, Nina-Beate Liabakk 1, Wei Wang 1, Magnar Bjørås 1,2 and Valentyn Oksenych 1,2,4,* 1 Department of Clinical and Molecular Medicine (IKOM), Norwegian University of Science and Technology, 7491 Trondheim, Norway; [email protected] (S.C.-Z.); [email protected] (C.H.); [email protected] (Ø.R.); [email protected] (A.S.); [email protected] (M.X.); [email protected] (R.G.-F.); [email protected] (Q.Z.); [email protected] (A.A.); [email protected] (J.W.); [email protected] (P.J.); [email protected] (N.-B.L.); [email protected] (W.W.); [email protected] (M.B.) 2 St. Olavs Hospital, Trondheim University Hospital, Clinic of Medicine, Postboks 3250, Sluppen, 7006 Trondheim, Norway 3 Molecular Biotechnology MS programme, Heidelberg University, 69120 Heidelberg, Germany 4 Department of Biosciences and Nutrition (BioNut), Karolinska Institutet, 14183 Huddinge, Sweden * Correspondence: [email protected]; Tel.: +47-913-43-084 These authors contributed equally to this work. y Received: 7 November 2019; Accepted: 26 November 2019; Published: 28 November 2019 Abstract: Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. The Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factor subunits X-ray repair cross-complementing group 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase 4 (Lig4). Accessory factors might be dispensable for the process, depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced a frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as the wild type controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development. Keywords: NHEJ; double-strand breaks; mouse model; lymphocyte; neurodevelopment 1. Introduction Non-homologous end-joining (NHEJ) is a molecular pathway that recognizes, processes, and repairs DNA double-strand breaks (DSBs) throughout the cell cycle [1]. Core NHEJ factors Ku70 and Ku80 form heterodimer (Ku) that is rapidly associated with the DSB sites facilitating recruitment of downstream factors including core x-ray cross-complementing 4 (XRCC4) and DNA ligase 4 (Lig4). XRCC4-like factor (XLF) is also a core factor that binds XRCC4 and stimulates Lig4-dependent DNA ligation. A number of accessory NHEJ factors are required for specific DNA end processing and DNA Biomolecules 2019, 9, 798; doi:10.3390/biom9120798 www.mdpi.com/journal/biomolecules Biomolecules 2019, 9, 798 2 of 13 complex stabilization, in other words, DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), nuclease Artemis and structural components, a paralogue of XRCC4 and XLF (PAXX), and modulator of retroviral infection (Mri) [2,3]. Mice lacking Ku70, Ku80, DNA-PKcs, or Artemis possess severe combined immunodeficient phenotype (SCID), while inactivation of both alleles of the Xlf gene results in 2–3-fold reduced B and T cell counts [1,4–7]. Mice lacking PAXX or Mri possess no or very modest phenotype due to functional redundancy with XLF [8–12]. In contrast, mice lacking either XRCC4 or Lig4 demonstrate p53- and Ku-dependent embryonic lethality, which correlates with massive neuronal apoptosis in the central nervous system [1,13–17]. Combined inactivation of Xlf and Dna-pkcs results in p53- and Ku70-dependent perinatal lethality in mice [10,18,19]. Moreover, deficiency or haploinsufficiency for Trp53 rescues synthetic lethality between Xlf and Paxx [10]. XLF is also functionally redundant in mouse development with Mri [20], recombination activating gene 2, RAG2 [21], and a number of DNA damage response (DDR) factors including Ataxia telangiectasia mutated (ATM) [6], histone H2AX [6,22], mediator of DNA damage checkpoint protein 1 (MDC1) [10], and p53-binding factor (53BP1) [7,23]. Development of B and T lymphocytes depends on programmed DSBs induced by RAG during the V(D)J recombination and NHEJ pathway,which is used for error-prone DNA repair [1]. Moreover, mature B cells replace constant regions of immunoglobulins during the somatic recombination process known as class switch recombination (CSR), when DSBs are initiated by activation-induced cytidine deaminase (AID) and Uridine-N-glycosylase (UNG), and NHEJ is used for DNA repair [1,24,25]. Furthermore, the NHEJ process is required for neurodevelopment by preventing neuronal apoptosis [1,26]. Mri was initially described as an open reading frame at human chromosome 7 (C7orf49), a factor reversing the resistance to retroviral infection in cell lines [27]. Mri was found to enhance NHEJ [28] and possess an N-terminal Ku-binding motif (KBM) [29]. Later, Mri/Cyren was suggested to inhibit NHEJ at telomeres during the S and G2 phases of the cell cycle [30], and finally confirmed to be a bona fide NHEJ factor, which is functionally redundant with XLF in mouse development including the V(D)J recombination and development of the central nervous system [20]. However, it was not clear whether XLF and Mri functionally overlap during the early stages of neurodevelopment (e.g., supporting proliferation and self-renewal of neuronal stem cells). Moreover, due to the lack of a viable mouse model deficient for both XLF and Mri, the impact of Mri on B and T lymphocyte development in vivo is not fully understood. Here, we introduced a frame-shift mutation to exon 2 of the murine Mri gene. By interbreeding / +/ +/+ heterozygous parents, we obtained Mri− −, Mri −, and Mri mice at nearly expected ratios. Mri-deficient mice possessed normal body size and number of B and T lymphocytes; however, / / we detected that stimulated primary mature Mri− − B cells had reduced levels of IgG1, and Mri− − neurospheres showed a reduced proliferation rate when compared to the Mri+/+ controls. 2. Materials and Methods 2.1. Mouse Models All experiments involving mice were performed according to the protocols approved by the Animal Resources Care Facility of Norwegian University of Science and Technology (NTNU, Trondheim, / +/ Norway). Ung− − mice were described previously [31]. Mri − mice were generated on request and described here for the first time. +/ 2.2. Generation of Mri − Mice / MRI-deficient (M− −) mice were generated through a CRISPR/Cas9 gene-editing approach in 2017 by Horizon Discovery (Saint Louis, MO, USA) upon request from the Oksenych group (IKOM, Faculty of Medicine and Health Science, NTNU, Trondheim, Norway). Single-guide RNA (sgRNA) GGG CTG TCA TCC AAG AGG GGA GG was designed to target exon 2 of the Mri gene in C57BL/6 mice. The 14 bp deletion resulted in a premature stop codon (Figure1A). Cas9 and sgRNAs were injected Biomolecules 2019, 9, 798 3 of 13 into single-cell fertilized embryos, which were then transferred back into pseudopregnant females for gestation. Live-born pups were screened for indel mutation by DNA sequencing. Homozygous pups +/ were used for back-crossing with wild type C57BL/6 mice. Heterozygous Mri − mice were obtained from Horizon Discovery. Biomolecules 2019, 9, x FOR PEER REVIEW 5 of 13 / / Figure 1. GenerationFigure of 1.Modulator Generation of Modulator of retrovirus of retrovirus infection infection−/− (−Mri−−/−() Mrimice. −(A−) T)op mice.. Schematic (A diagram) Top. of Schematic diagram murine Mri locus indicating the frame-shift mutation in the exon 2, induced by the single guide RNA of murine Mri locus(sgRNA indicating) and resulting the in a frame-shift14 bp deletion. (Bottom mutation) Resulting inMri−/− the locusexon lacking 2 part, induced of the exon 2 by. the single guide (B) Top. Polymerase chain reaction (PCR)-based genotyping strategy reveals the Mri WT/ allele (428 RNA (sgRNA) and resulting in a 14 bp deletion. (Bottom) Resulting Mri− − locus lacking part of the bp) and Mri null allele (414 bp). (Bottom) WT gene validation PCR revealed the Mri wild type allele exon 2.(B) Top. Polymerase(234 bp). (C) Analyses chain of reaction 140 pups born (PCR)-based from Mri+/- parents genotyping revealed expected strategygenetic distribution reveals of the Mri WT allele (428 bp) and Mri nullMri allele+/+ (29), Mri (414+/− (75) bp)., and Mri (Bottom)−/− (36) mice, WTwhich geneis close to validation the Mendelian PCRdistribution revealed 1:2:1. (D) theBody Mri wild type allele weight of six to eight week old Mri+/+ mice (n = 6) was not distinguishable from Mri−/− mice (n = 7), p = +/- (234 bp). (C) Analyses0.4242. of(E) 140The weight pups of bornspleens fromisolated Mrifrom Mri+/+parents (n = 8) and revealed Mri−/− mice (n expected = 11) was not genetic distribution +/+ +/significantly different, p = 0.8551./ Spleen size in immunodeficient Dna-pkcs−/− mice (n = 10) was reduced of Mri (29), Mri − (75), and Mri− − (36) mice, which is close to the Mendelian distribution 1:2:1.